The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle.

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.

Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Genistein improves renal disease in a mouse model of nephropathic cystinosis: a comparison study with cysteamine.

Cysteamine is currently the only therapy for nephropathic cystinosis. It significantly improves life expectancy and delays progression to end-stage kidney disease; however, it cannot prevent it. Unfortunately, compliance to therapy is often weak, particularly during adolescence. Therefore, finding better treatments is a priority in the field of cystinosis. Previously, we found that genistein, an isoflavone particularly enriched in soy, can revert part of the cystinotic cellular phenotype that is not sensitive to cysteamine in vitro. To test the effects of genistein in vivo, we fed 2-month-old wild-type and Ctns-/- female mice with either a control diet, a genistein-containing diet or a cysteamine-containing diet for 14 months. Genistein (160 mg/kg/day) did not affect the growth of the mice or hepatic functionality. Compared with untreated mice at 16 months, Ctns-/- mice fed with genistein had lower cystine concentrations in their kidneys, reduced formation of cystine crystals, a smaller number of LAMP1-positive structures and an overall better-preserved parenchymal architecture. Cysteamine (400 mg/kg/day) was efficient in reverting the lysosomal phenotype and in preventing the development of renal lesions. These preclinical data indicate that genistein ameliorates kidney injury resulting from cystinosis with no side effects. Genistein therapy represents a potential treatment to improve the outcome for patients with cystinosis.

Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light-responsive miR-211 controls lysosomal biogenesis at the beginning of light-dark transitions in the RPE by targeting Ezrin, a cytoskeleton-associated protein essential for the regulation of calcium homeostasis. miR-211-mediated down-regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light-mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR-211-/- mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR-211-/- phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.

Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin.

Mutant Z alpha-1 antitrypsin (ATZ) accumulates in globules in the liver and is the prototype of proteotoxic hepatic disease. Therapeutic strategies aiming at clearance of polymeric ATZ are needed. Transient receptor potential mucolipin-1 (TRPML1) is a lysosomal Ca2+ channel that maintains lysosomal homeostasis. In this study, we show that by increasing lysosomal exocytosis, TRPML1 gene transfer or small-molecule-mediated activation of TRPML1 reduces hepatic ATZ globules and fibrosis in PiZ transgenic mice that express the human ATZ. ATZ globule clearance induced by TRPML1 occurred without increase in autophagy or nuclear translocation of TFEB. Our results show that targeting TRPML1 and lysosomal exocytosis is a novel approach for treatment of the liver disease due to ATZ and potentially other diseases due to proteotoxic liver storage.

Effect of post-printing curing time on cytotoxicity of direct printed aligners: A pilot study.

INTRODUCTION: The aim of this in vitro study was to examine the potential impact of different curing times of 3D-printed orthodontic aligners on their cytotoxicity. METHOD: Some 60 samples of aligner material were directly 3D printed using Tera Harz TC-85 DAC resin and randomly allocated to three different curing time groups (14, 24 and 50 min). Zendura FLX samples were used as control. The samples were incubated in saliva for 14 days, and then the supernatant was collected. Human gingival fibroblasts (HGF-1)-CRL2014 were used to evaluate potential cytotoxicity. Furthermore, HGF-1 cells were plated on the samples as well as on a glass control sample. After 72 h of growth, their viability was tested. RESULTS: Compared with the glass, only the 50-min curing time markedly reduced fibroblast cell growth. Additionally, a negative linear trend was observed between curing time and fibroblast growth. In comparison with the aligner control group, all samples, including the aligner control samples, exhibited a significant reduction in the viability of human fibroblasts when exposed to saliva. CONCLUSIONS: 3D directly printed aligners showed a cytotoxic effect similar to that of thermoformed conventional aligners in terms of fibroblasts growth. A linear trend was found between curing time and cells growth, indicating that directly printed aligners could exhibit higher cytotoxicity if exposed to a longer curing time. This dependence on curing time underscores the importance of following a strict manufacturing process.

The TRAPP complex mediates secretion arrest induced by stress granule assembly.

The TRAnsport Protein Particle (TRAPP) complex controls multiple membrane trafficking steps and is strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified the TRAPP complex as a component of a branch of the integrated stress response that impinges on the early secretory pathway. The TRAPP complex associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and arrest of ER export. The relocation of the TRAPP complex and COPII to SGs only occurs in cycling cells and is CDK1/2-dependent, being driven by the interaction of TRAPP with hnRNPK, a CDK substrate that associates with SGs when phosphorylated. In addition, CDK1/2 inhibition impairs TRAPP complex/COPII relocation to SGs while stabilizing them at ER exit sites. Importantly, the TRAPP complex controls the maturation of SGs. SGs that assemble in TRAPP-depleted cells are smaller and are no longer able to recruit RACK1 and Raptor, two TRAPP-interactive signaling proteins, sensitizing cells to stress-induced apoptosis.

Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and Collagen quality control.

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.